In many species of higher animals, immunoglobulins (Ig) can be divided into five different classes: IgG, IgA, IgM, IgD, and IgE. These classes are different in size, charge, amino acid composition, sugar content, and the like. Among these classes, IgM makes up approximately 10% of whole plasma immunoglobulin. IgM is a major component of early-stage antibodies produced against cell membrane antigens that are of complex antigenicity, infectious microorganisms, and soluble antigens.
In general, human IgM has a pentamer structure in vivo. The five subunits, which constitute the pentamer structure of IgM, have a four-chain structure similar to IgG. The μ chain, the H (heavy) chain of IgM, is different from the γ chain, the H chain of IgG, with respect to the amino acid sequences, and additionally, they are different as follows:                The μ chain has one more domain in its constant region than the γ chain.        The μ chain has four more oligosaccharide chains than the γ chain.        IgM has a polypeptide chain called J chain, which is not found in IgG. The J chain is considered to facilitate polymerization of μ chains before IgM is secreted from antibody-producing cells.        
In recent years, advances in monoclonal antibody technology and recombinant DNA technology have enabled mass production of pure immunoglobulin. Moreover, genetic engineering technology has allowed the production of chimeric and humanized antibodies. A chimeric antibody is an antibody which has a structure where a variable region has been changed to another variable region of a different species origin. For example, a “chimeric antibody” which has a variable region of a non-human animal species and a constant region of a human antibody is known (Reference 1/Proc. Natl. Acad. Sci. U.S.A, (1984) 81:6851). A humanized antibody made by transplanting complementarity determining regions (CDRs) from an animal species into human immunoglobulin is also known (Reference 2/Nature (1986) 321:521).
The anti-CD20 human chimeric antibody, Rituxan® (IDEC), and the anti-HER2/neu humanized antibody, Herceptin® (Genentech), are specific examples of anti-tumor antibodies. These antibodies have already passed clinical trials and have been approved for distribution. Antibody-dependent cellular mediated cytotoxicity (hereinafter referred to as ADCC activity) and complement-dependent cytotoxicity (hereinafter referred to as CDC activity) are known as effector functions of IgG and IgM. Since IgM has a CDC activity higher than that of IgG, it can be used as an anti-tumor antibody with the CDC activity as a main drug activity. However, unlike IgG, IgM forms a polymer as mentioned above. For this reason, the industrial scale production of recombinant IgM has been considered to be difficult.
Several production systems for IgM recombinants using non-lymphoid cells have been reported. For example, introducing the genes of IgM H and L (light) chains into C6 glioma cells, CHO cells, or HeLa cells successfully resulted in the formation of polymer; however, the yield from the CHO cells was very low (Reference 3/EMBO J. (1987) 9; 2753) (Patent document 1/W089/01975). Moreover, IgM-producing CHO cell lines were obtained by incorporating IgM H and L chains into separate expression vectors and co-expressing them (Reference 4/J. Immunol. (1990) 145; 3011) (Reference 5/Human Antibodies (1997) 8; 137). These reports also showed that the recombinant IgM produced by the CHO cells formed a polymer, but they failed to reveal a ratio of pentamer and hexamer or the like.
The main reason that a polymeric structure of IgM recombinant has not been developed is that no analytical technique has been established. Specifically, the polymeric structure of IgM cannot be correctly analyzed using known analytical methods for immunoglobulin,. For example, gel electrophoresis, such as SDS-PAGE, is known as a technique for separating and identifying proteins. However, the IgM macromolecule has a molecular weight of approximately one million. It is therefore difficult to quantitatively analyze the polymeric structure (pentamer and hexamer) using conventional means.
In a reported technique for analyzing the IgM polymeric structure, non-reducing SDS-PAGE was performed using RI-labeled IgM (Reference 6/J. Immunol. (1994) 152; 1206). However, to develop IgM as a drug, it is necessary to develop a technique with which the polymeric structure of IgM can be analyzed in manufacturing process. Specifically, analysis of the polymeric structure of IgM is required in all the steps of manufacturing, including selection of producing cells, monitoring of cell culture, purification, primary drug manufacturing, and pharmaceutical preparation. The use of RI, however, is not practical for assessment of all these steps.    [Reference 1] Proc. Natl. Acad. Sci. U.S.A, (1984) 81:6851    [Reference 2] Nature (1986) 321:521    [Reference 3] EMBO J. (1987) 9;2753    [Reference 4] J. Immunol. (1990) 145;3011    [Reference 5] Human Antibodies (1997) 8;137    [Reference 6] J. Immunol. (1994) 152;1206    [Patent document 1] WO89/01975